Isolation and characterization of neurokinin A, neurokinin A(3-10) and neurokinin A(4-10) from a neutral water extract of a metastatic ileal carcinoid tumour

A metastasis to the right liver lobe of an argyrophil/argentaffin midgut carcinoid tumour in a patient with the classical carcinoid syndrome was examined for the presence of tachykinins other than substance P, using a specific antiserum. The extract was initially purified using SepPak cartridges, and subsequently subjected to cation-exchange chromatography on SP Sephadex C-25 which separated the immunoreactive material into two main components (components I and II). Both were further purified by anion-exchange chromatography on DEAE-Sephadex A-25, and by reverse-phase fast protein liquid chromatography. Component II was identified as neurokinin A by its immunochemical and chromatographic properties and amino acid sequence analysis. Component I consisted of two molecular forms which were identified as neurokinin A(3-10) and neurokinin A(4-10) by amino acid sequence analysis. The tumour tissue contained only small amounts of the eledoisin-like peptide that has earlier been demonstrated in mammalian tissues. Although this component behaved like the nonmammalian peptide eledoisin on reverse-phase HPLC and on reverse-phase ion-pair chromatography, eledoisin-specific antiserum E2 indicated that eledoisin-like peptide is not identical to eledoisin. Neurokinin A in carcinoid tumours has an N-terminal heterogeneity; this multiplicity constitutes a further support for the hypothesis that carcinoid tumours produce a number of tachykinins which may be present in different relative amounts in individual patients and may contribute to the individual differences in symptomatology.     

Changes in H1 complement in differentiating rat-brain cortical neurons

Neuronal nuclei have a low H1 content. A stoichiometry of 0.47 molecule/nucleosome, on average, is calculated for rat brain cortical neurons by comparing its H1 content with that of liver nuclei. The H1 fraction of rat cerebral cortex neurons has been resolved into five subtypes, H1a--e, that have the same mobility as the unphosphorylated H1 forms of other rat tissues. The subtypes H1a--d decay exponentially during postnatal development and are substituted to different extents by H1e. The higher replacement rate is shown by H1a with an apparent half-lifetime of about 5 days. The corresponding values for H1b, H1c and H1d are 11, 21 and 15 days. Several conclusions can be drawn from the observation of postnatal changes in H1 subtype proportions. The low H1 content of neuronal nuclei does not imply the presence of notable peculiarities in subtype composition or in subtype substitution pattern. There is turnover of H1 in differentiating neurons once cell proliferation and DNA replication have ceased. The relative rates of synthesis and/or degradation of the subtypes differ in germinal cells and in neurons. Comparison with previous results on H1 degrees accumulation also shows that in cortical neurons the regulation of the subtypes H1a--e differs from that of H1 degrees.     

Cloning and sequencing of a rat CuZn superoxide dismutase cDNA. Correlation between CuZn superoxide dismutase mRNA level and enzyme activity in rat and mouse tissues

The molecular cloning and nucleotide sequence of a cDNA clone (pR SOD) for rat CuZn superoxide dismutase (CuZnSOD) is reported. Nucleotide sequence homology with human superoxide dismutase is 86% for the coding region and 71% for the 3' untranslated region. The deduced amino acid sequence is given and the homologies with the sequences reported for other species are presented. Northern blot analysis of total RNA from various rat and mouse tissues and from two mouse cell lines show that pR SOD hybridizes with one mRNA species of about 0.7 kb. The amount of CuZnSOD mRNA in each tissue, measured by densitometry of the Northern blot autoradiograms, correlates with the enzymatic activity based on protein content. These results indicate that the control of CuZnSOD activity in mammalian tissues is largely dependent on the regulation of CuZnSOD mRNA levels. In human liver, fibroblasts and FG2 hepatoma cells, two CuZnSOD mRNAs (0.7 kb and 0.9 kb) are observed. The level of CuZnSOD mRNA in FG2 is 25% that of the liver and four times more abundant than in fibroblasts.     

Unimpaired coupling of phosphorylated, desensitized beta-adrenoceptor to Gs in a reconstitution system

Heterologous desensitization of turkey erythrocyte beta-adrenoceptors correlates with receptor phosphorylation and impaired receptor-Gs coupling, as assessed by fusion of purified desensitized receptors with X. laevis erythrocytes [(1984) Science 225, 837-840]. We have purified beta-receptors from desensitized and untreated turkey erythrocytes and have compared the abilities of these two receptors to couple with pure turkey erythrocyte Gs in a reconstituted system. Functional receptor-Gs coupling was assessed by measuring hormone-dependent Gs activation by GTP gamma S and GTPase activity. While in membranes prepared from desensitized cells, receptor-Gs coupling was clearly reduced, this effect was absent when coupling of purified desensitized receptor was measured. We conclude that covalent modification by phosphorylation does not fully explain the functional uncoupling at the membrane level.     

Characterization of the nucleotide tight-binding sites of the isolated mitochondrial F1-ATPase

The properties of the nucleotides tightly bound with mitochondrial F1-ATPase were examined. One of three bound nucleotide molecules is localized at the site with Kd approximately 10(-7) M and released with koff approximately 0.1 s-1. The second nucleotide molecule is bound with the enzyme with Kd approximately 10(-8) M and koff for its dissociation is 3 X 10(-4) s-1. The third is never released even in the presence of 1 mM ATP or ADP. The last two nucleotides are believed to be bound at the noncatalytic sites of F1-ATPase. Pyrophosphate promotes liberation of two releasable nucleotide molecules, decreasing the affinity of the enzyme to AD(T)P. From the results obtained it follows that the only suitable criterion for localization of the nucleotide at the F1-ATPase catalytic site is the high rate (koff greater than or equal to 0.1 s-1) of its spontaneous release.     

On secondary hyperparathyroidism in orang-utans, Pongo satyrus (Linnaeus, 1758)

Orang-utan crania with alterations in bone structure which could be determined morphologically were studied and evaluated. The alterations in bone structure were referred to in the literature up to about 1939 as "rickets"; when vitamin D was given to the animals, the alterations diminished, until they were almost unnoticeable. From about 1941/52, the alterations were diagnosed as "Morbus Paget". Research on orang-utan crania has become possible through comparison of a larger number of single symptoms, occurring in a number of individuals. Out of a larger sample, the study was carried out on 5 individuals, showing these alterations in varying degrees. The individuals also covered various age groups, both sexes and both subspecies of orang-utans. The findings permit a diagnosis of secondary (or tertiary) hyperparathyroidism (= Morbus Engel-von Recklinghausen = fibrous cystic osteitis). The study also showed that orang-utans fall prey to Morbus Engel-von Recklinghausen in a shorter period and suffer more severely than humans. The frequency of orang-utans suffering from this disease, which are kept captive in zoos, is statistically far higher than the occurrence in humans. Orang-utans living under natural conditions do not suffer from the disease at all; according to the study of 500 animals. The authors also believe that there is a psychogenic basis for the occurrence of Morbus Engel-von Recklinghausen in zoo animals; psychological conditions such as apathy, disinterest, etc. are part of the illness, and these symptoms are also shown by animals having no outward signs of fibrous cystic osteitis. The authors believe that improved prophylaxis of orang-utans in zoos would lead to discovery of Morbus Engel-von Recklinghausen in early stages, and allow early therapy to arrest the disease. This is necessary for preservation of the species.     

Characterization of a heterogeneous camel milk whey non-casein protein

A milk protein, occurring in the whey fraction, has been characterized from camel milk. Determination of the primary structure reveals the existence of two related types of chain with residue differences in at least the N-terminal region. A fragment representing an N-terminal part of the protein was also recovered (heterogeneous at the same positions). The absence of cysteine residues in the protein shows that no disulphide bridges are present. The pattern of fragments and a parent protein resembles that for casein and its fragments, showing that fragments and a multiplicity of forms may be typical for different milk proteins.     

Comparative study of the transformation of various thymidine kinase-deficient human and animal cell lines with the thymidine kinase gene of the Herpes simplex virus

The comparative study of transformation of four thymidine kinase deficient cell lines (mouse mammary carcinoma cell line FS tk-; rat cell line Rat-2tk-; mouse cell line Ltk-, clone D1; human cell line 143tk-) with the thymidine kinase cloned gene of Herpes simplex virus 1 was undertaken. The differences in efficiency and optimal conditions of transformation were shown for these cell lines. The advantages and disadvantages of the cell systems examined for the use in experiments for transformation and cotransformation of cultured cells with isolated genes are discussed.     

Quantitative analysis of C-segments of chromosomes 1, 9, 16 and Y in couples with reproductive disorders

A comparative analysis of structural variability of C-bands on chromosomes 1, 9, 16 and Y was conducted in 50 phenotypically normal adults and 25 couples with repeated spontaneous abortions. Reduction of both the total amount of heterochromatin in the cell and the lengths of these regions on chromosomes 1, 9, and 16 is revealed in the group of pathology. No differences were found in the lengths of C-bands on Y chromosome.     

Determination of the number of tissue compatibility genes in the carp using the transplantation test in the third generation of induced gynogenesis

Transplantations have been carried out of anal fin in carp individual progeny of the third consecutive generation of induced gynogenesis. The part of grafts accepted made 19.4 +/- 5.0% (71 transplantations have been carried out). With the data obtained, an approximate quantity of histocompatible genes has been determined in carp which amounted to 32. Calculation has been done on the basis of the assumption that the part of histocompatible genes being in homozygous state in the given progeny equals the inbreeding coefficient of this progeny. Inbreeding coefficients have been calculated using experimental data of other authors.